To reduce costs of NGS, libraries are often prepared using barcodes or adapters to facilitate multiplexing (combining) of samples. These barcodes contain index sequences, short sequences of 6 – 12 bases that are incorporated into each library to allow several libraries to be pooled and sequenced together on a single flow-cell of a sequencer. When barcoded NGS libraries are sequenced, each cluster generated on an Illumina® sequencer includes its own unique index sequence, which is then associated with a specific library.

High levels of multiplexing can involve use of large sets of barcodes. With high level multiplexing, at any position of the index all 4 bases can be found, ensuring sufficient diversity.

Low-plex multiplexing (i.e. mixing up to ~ 12 samples) is often chosen as a reasonable compromise between low cost/low per sample coverage, and high cost/high coverage. When using low-level multiplexing, due to the limited number of barcodes involved, it is possible that the index sequences chosen could lack sufficient diversity to avoid “registration failure” on an Illumina® sequencer.

Registration failure could occur if the color balance was not maintained between the red and green lasers (used to sequence A/C bases and G/T bases, respectively). Barcode sequences for low-level multiplexing need to be chosen such that each position of the barcode will result in signal in both the red and green color channels. In other words, each position in the set of index sequences needs to include at least one A or C base, at least one G or T base, and ideally an equal balance of both.

Examples of acceptable combinations of barcodes for 2-way multiplexing:

Every position in each pair has at least one A/C and one G/T

NGS Barcodes

Examples of unacceptable barcode combinations for 2-way multiplexing:

In this example, the first pair lacks G/T at positions 2 and 3 and lacks A/C at position 6; second pair lacks G/T at first position, lacks C/A at fourth and fifth positions

NGS Barcodes

With these considerations in mind, PerkinElmer recommends the use of specific barcode combinations for low-level multiplexing. These low-level multiplexing recommendations can be found in Appendix A in all of our barcode manuals. Note that recommendations are given for low-plex barcode combinations even for our larger barcode sets, to provide flexibility for use in situations where low-plex multiplexing is desirable. The NEXTFLEX® Unique Dual Index barcodes were designed to allow a broad range of multiplexing – users can multiplex anywhere between two samples and 1,536 samples in a single sequencing run.

Read our Next Tech Tip on NGS Multiplexing: Multiplexing Libraries Containing Indexes of Different Lengths

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